“Pulmonary surfactant” or “lung surfactant” (LS) is a mixture of proteins and lipids that coats the internal surfaces of healthy mammalian lungs and enables normal breathing [1]. By virtue of its unique surface-active properties, lung surfactant markedly decreases the surface tension at the air-liquid interface of the myriad tiny air-sacs that perform gas exchange within the lung (“alveoli”), reducing the pressure required for alveolar expansion and decreasing the work of breathing [2, 3]. Lung surfactant also stabilizes the alveolar network upon exhalation, preventing collapse [3, 4].
Natural lung surfactant is composed of 90-95% lipids and 5-10% protein [5, 6, 7]. Both protein and phospholipid fractions play critical roles in physiological surface activity [8]. Phosphatidylcholine (PC) variants are the most abundant components, making up 70-80% of the lipid fraction. 50-70% of the PC molecules are saturated and dipalmitoylated (DPPC). Anionic phosphatidylglycerol (PG) accounts for 8%, and other lipids as well as cholesterol are present in minor amounts [5].
In vitro and in vivo biophysical experiments have shown that the most critical lipid molecules for surface tension reduction are DPPC and PG [6, 7]. However, lipid mixtures alone are ineffective as lung surfactant replacements, because under physiological conditions and in the absence of “spreading agents,” DPPC and PG do not adsorb to the air-liquid interface quickly, nor can they be respread rapidly as alveolar surface area changes cyclically [9]. Instead, a unique class of protein surfactants function as spreading agents.
Four surfactant-associated proteins are present with phospolipids on the alveolar hypophase: SP-A, SP-B, SP-C, and SP-D [10]. These fall into two major subgroups: hydrophilic surfactant proteins SP-A and SP-D, and hydrophobic surfactant proteins SP-B and SP-C. SP-A and SP-D control surfactant metabolism, and also play important immunological roles as a defense against inhaled pathogens [11]. But for therapeutic lung surfactant replacements, it is the biophysical properties of surfactant—as they affect the mechanical properties of the lung—that are important for the treatment of respiratory distress. Neither SP-A nor SP-D is responsible for the surface tension-lowering properties of surfactant [6], so they are typically omitted from surfactant replacements.
Surfactant-associated proteins are required for proper functioning of lung surfactant [8], and it is the small hydrophobic proteins SP-B and SP-C that enable low surface tensions on the alveolar hypophase, endowing a proper dynamic behavior of lipid monolayers [12-14]. SP-B and SP-C interact non-synergistically with lipids to enable easy breathing [15]. In vivo rescue experiments with premature rabbits [16], in vivo blocking of SP-B with monoclonal antibodies [17], and studies with genetically-engineered SP-B-deficient mice [18] all confirm the necessity of SP-B and SP-C proteins for functioning of lung surfactant in vivo [8]. Both facilitate rapid adsorption of phospholipids to an air/water interface, allowing rapid re-spreading of phospholipids as alveoli expand and contract. Both influence the monolayer's phase behavior, and reduce surface tension on alveoli at expiration to <1 mN/m [14, 19].
Neonatal Respiratory Distress Syndrome (NRDS) is a leading cause of infant mortality in the United States [6]. In the absence or dysfunction of pulmonary surfactant, mammalian lungs are incompliant and vulnerable to alveolar collapse upon expiration, due to excessive surface tension forces. Preterm infants who have gestated <29 weeks have not yet begun to secrete lung surfactant into alveolar spaces [20] and suffocate after delivery without surfactant replacement therapy. Hence, it is standard care for infants with NRDS (given prophylactically for infants born before 28 weeks gestation), and is expected to gain clinical significance for “acute RDS” (ARDS) in adults and children [6].
Adults and children would also benefit from an effective, non-immunogenic, bioavailable, and less expensive synthetic surfactant replacement. Dysfunction of surfactant is a major contributor to the lethal ARDS, which can occur in adults and children after shock, bacterial sepsis, hyperoxia, near drowning, or aspiration [6]. ARDS is a leading cause of death in intensive care units, and as yet has no generally effective, economically viable treatment [7]. The dysfunction of lung surfactant in adults and children most typically results from the encroachment of blood serum or other foreign fluids into the lungs. Serum proteins disrupt and inhibit the spreading of natural surfactant by poorly-understood biorecognition and bioaggregation mechanisms [9, 21]. Lung surfactant replacement therapy was investigated for the treatment of adult and child ARDS [22-25]. But the large doses necessary for adults make this potentially useful treatment far too expensive [26].
Academic and industrial research have resulted in the commercialization of several functional lung surfactant replacements, but the material is quite expensive ($1000 per 1.2-mL dose) and different formulations give highly variable results [27, 28]. Animal-derived surfactants are most expensive, and work best to restore lung function quickly, but raise purity and immunological concerns [27]. If infants with NRDS survive surfactant replacement therapy (they need up to four doses every 6-8 hours after birth), they begin to secrete their own pulmonary surfactant within 96 hours [5].
Currently, the two classes of lung surfactant replacements commercially available for the treatment of respiratory distress syndrome (RDS) are “natural” and “synthetic.” “Natural surfactant replacements” are prepared from animal lungs by lavage or extraction with organic solvents, and purified by chromatography [5, 6, 26]. A number of animal-derived surfactant replacements are FDA-approved [29-32]. “Synthetic surfactant replacements” are by definition protein-free, and are made from synthetic phospholipids with added chemical agents (lipids or detergents) to facilitate adsorption and spreading [33, 34]. These protein-free synthetic formulations do not work well, and have fallen out of common use.
A third, not-yet-commercially-available class of formulations is the “biomimetic lung surfactants.” Biomimetic surfactants are designed to mimic the biophysical characteristics of natural lung surfactant while not sharing its precise molecular composition. These formulations contain synthetic phospholipid mixtures in combination with recombinantly-derived or chemically-synthesized peptide analogs to SP-B and/or SP-C [7].
Since biomimetic surfactant formulations are not available, doctors must choose between animal-derived or synthetic surfactant replacements [27, 35, 36]. Despite worries about the possible contamination of animal-derived surfactants with animal viruses, and problems with rapid surfactant biodegradation resulting in a need for multiple doses [27], most doctors favor animal-derived formulations [6]. Current synthetic formulations (although safer, generally effective, and less expensive than natural surfactants) [27] have inferior in vivo efficacy (saving 1 fewer infant per 42 treated [27, 36]), primarily because better analogs for the SP-B and SP-C proteins are needed.
Bovine and porcine SP are ˜80% homologous to human SP, and are recognized as foreign by the immune system even in some infants [17, 37, 38]. Antibodies that develop to these homologous SP sequences have the potential to inactivate natural human SP and lead to respiratory failure. This has not yet been found to occur in newborns [5, 6], but for adults with ARDS, auto-antibodies could be a serious problem [27]. Surfactant replacement therapy in premature infants has a high failure rate (˜65% of infants die or develop chronic lung disease (bronchopulmonary dysplasia, BPD) after therapy) [27].
When human medicines are extracted from animals it is impossible to eliminate the chance of cross-species transfer of antigenic or infectious agents or unforeseeable biological contamination [39]. Synthetic, biomimetic surfactants obviate these risks, and may also offer greater bioavailability (fewer doses, hence lower cost) and less liability to inhibition. Synthetic surfactants must be improved until efficacy for RDS rescue therapy with synthetics matches that of natural surfactant.
To obviate the need for animal-derived medicines, several groups have undertaken de novo chemical synthesis of truncated peptide mimics of SP-B and SP-C for surfactant preparations [7]. The majority of these synthetic, biomimetic polypeptides have been biophysically functional in vitro and in vivo (i.e., they have been successful to some degree in promoting achievement of low surface tensions and facilitating rapid re-spreading of surfactant lipids, allowing the rescue of premature animals with RDS). Several workers, including Kang [40], Bruni [41], and Lipp [42-44], have made and tested SP-B fragments. All succeeded in making biophysically-active SP-B analogs. Interestingly, a 25-residue peptide from the amino-terminus of SP-B seems to capture the surface-active properties of full-length SP-B [42]. Fujiwara [45] and Notter [46] made shortened mimics of SP-C, while Wang [46] made full-length, palmitoylated SP-C peptide and reported that acylation of cysteines is critical for SP-C's biophysical function. Takei et al. [45] omitted the palmitoyl groups and found that shortened SP-C peptide mimics (residues 5-32) retain “full biophysical activity” in vitro and in vivo. What is striking about these studies is that many groups have made peptide mimics of SP, and all were successful to some degree. This provides strong evidence of the tolerance of this system for slight variations in SP analogs—to be expected since they interact primarily with lipids, which is likely an interaction of a much less specific nature than many biomolecule interactions.
As indicated by the notations herein, these and other aspects of the prior art as related to an understanding of this invention can be found in the following:
1. Pattle, R. E., Properties, function, and origin of the alveolar lining layer. Nature, 1955, 175: p. 1125-1126.
2. Clements, J. A., Surface tension of lung extracts. Proc. Soc. Exp. Biol. Med., 1957. 95: p. 170-172.
3. Clements, J. A., E. S. Brown, and R. P. Johnson, Pulmonary surface tension and the mucus lining of the lungs: Some theoretical considerations. J. Appl. Physiol., 1958. 12: p. 262-268.
4. Putz, G., et al., Comparison of captive and pulsating bubble surfactometers with use of lung surfactants. J. Appl. Physiol., 1994. 76: p. 1425-1431.
5. Creuwels, L. A. J. M., M. G. van Golde, and H. P. Haagsman, The pulmonary surfactant system: Biochemical and clinical aspects. Lung, 1997. 175: p. 1-39.
6. Notter, R. H., and Z. Wang, Pulmonary surfactant: Physical chemistry, physiology, and replacement. Reviews in Chemical Engineering, 1997. 13: p. 1-118.
7. McLean, L. R., and J. E. Lewis, Biomimetic pulmonary surfactants. Life Sciences, 1995. 56: p. 363-378.
8. King, R. J., and J. A. Clements, Surface active materials from dog lung. II. Composition and physiological correlations. Am. J. Physiol., 1972. 223: p. 715-726.
9. Cockshutt, A., D, Absolom, and F. Possmayer, The role of palmitic acid in pulmonary surfactant: Enhancement of surface activity and prevention of inhibition by blook proteins. Biochim, Biophys. Acta, 1991. 1085: p, 248-256.
10. Johansson, J., T. Curstedt, and B. Robertson, The proteins of the surfactant system. Eur. Respir. J., 1994. 7: p. 372-391.
11. Khoor, A., et al., Developmental expression of SP-A and SP-A mRNA in the proximal and distal epithelium in the human fetus and newborn. J. Histochem. Cytochem, 1993. 41: p. 1311-1319.
12. Hall, S. B., et al., Importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants. Am. Rev. Respiratory Disorders, 1992. 145: p. 24-30.
13. Goerke, J., Pulmonary surfactants-Physicochemical aspects. Current Opinion in Colloid & Interface Science, 1997. 2: p. 526-530.
14. Wang, Z., S. B. Hall, and R. H. Notter, Roles of different hydrophobic constituents in the adsorption of pulmonary surfactant. Journal of Lipid Research, 1996. 37: p. 790-798.
15. Wang, Z., et al., Differential activity and lack of synergy of lung surfactant proteins SP-B and SP-C interactions. Journal of Lipid Research, 1996. 37: p. 1749-1760.
16. Rider, E. D., et al., Treatment responses to surfactants containing natural surfactant proteins in preterm rabbits. Am. Rev. Respir. Dis., 1993. 147: p. 669-676.
17. Robertson, B., et al., Experimental neonatal respiratory failure induced by a monoclonal antibody to the hydrophobic surfactant-associated protein SP-B. Pediatr. Res., 1991. 30: p. 239-243.
18. Tokeida, K., et al., Pulmonary dysfunction in neonatal SP-B-deficient mice. Am. J. Physiol., 1997. 273: p. L875-L882.
19. Taneva, S. and K. M. W. Keogh, Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface. I1I. Proteins SP-B plus SP-C with phospholipids in spread monolayers. Biophys. J., 1994. 66: p. 1158-1166.
20. Goerke, J. and J. A. Clements, Alveolar surface tension and lung surfactant, in Handbook of Physiology: The Respiratory System—Control of Breathing. 1986, American Physiology Society: Bethesda, Md. p. 247-261.
21. Jobe, A., et al, Permeability of premature lamb lungs to protein and the effect of surfactant on that permeability. J. Appl. Physiol., 1983. 55: p. 169-176.
22. Gregory, T. J., et al., Survanta supplementation in patients with acute respiratory distress syndrome (ARDS). Am. J. Resp. Cell. Mol. Bio., 1994. 149: p. A567.
23. Spragg, R. G., et al., Acute effects of a single dose of porcine surfactant on patients with adult respiratory distress syndrome. Chest, 1994. 105: p. 195-202.
24. Hafner, D., et al., Dose response comparisons five lung surfactant factor (LSF) preparations in an animal model of adult respiratory distress syndrome (ARDS). Br. J. Pharmacol., 1995. 116: p. 451-458.
25. Willson, D. F., et al., Calf's lung surfactant extract in acute hypoxemic respiratory failure in children. Crit. Care Med., 1996. 24: p. 1316-1322.
26. Kattwinkel, J., Surfactant: Evolving issues. Clinics in Perinatology, 1998. 25: p. 17-32.
27. Whitelaw, A., Controversies: Synthetic or natural surfactant treatment for respiratory distress syndrome? The case for synthetic surfactant. J. Perinat. Med., 1996. 24: p. 427-435.
28. Halliday, H. L., Synthetic or natural surfactants. Acta Paediatr., 1997. 86: p. 233-7.
29. Hoekstra, R. E., et al., Improved neonatal survival following multiple doses of bovine surfactant in very premature neonates at risk of respiratory distress syndrome. Pediatrics, 1991. 88: p. 19-28.
30. Gortner, L. A., A multicenter randomized controlled trial of bovine surfactant for prevention of respiratory distress syndrome. Lung, 1990. 168 (Suppl): p. 864-869.
31. Kendig, J. W., et al., A comparison of surfactant as immediate prophylaxis and as rescue therapy in newborns of less than 30 weeks gestation. N. Engl. J. Med., 1991. 324: p. 865-871.
32. Collaborative European Multicenter Study Group. Surfactant replacement therapy in severe neonatal respiratory distress syndrome: An international randomized clinical trial. Pediatrics, 1988. 82: p. 683-691.
33. Morley, C. J., et al., Dry artificial lung surfactant and its effect on very premature babies. Lancet, 1981. i: p. 64-68.
34. Phibbs, R. H., et al., Initial clinical trial of Exosurf, a protein-free synthetic surfactant, for the prophylaxis and early treatment of hyaline membrane disease. Pediatrics, 1991. 88: p. 1-9.
35. Zetterström, R., Surfactant therapy: Clinical implications. Acta Paediatr., 1996. 85: p. 641-641.
36. Halliday, H. L., Controversies: Synthetic or natural surfactant. The case for natural surfactant. J. Perinat. Med., 1996. 24: p. 417-426.
37. Strayer, D. S., et al., Surfactant anti-surfactant immune complexes in infants with respiratory distress syndrome. Am. J. Pathology, 1986. 122: p. 353-362.
38 Chida, S., et al., Surfactant proteins and anti-surfactant antibodies in sera from infants with respiratory distress syndrome. Pediatrics, 1991. 88: p. 84-89.
39. Long, W., Synthetic surfactant. Seminars in Perinatology, 1993. 17: p. 275-284.
40. Kang, J. H., et al., The relationships between biophysical activity and the secondary structure of synthetic peptides from the pulmonary surfactant protein SP-B. Biochem. and Molec. Biol, Intl., 1996. 40: p. 617-627.
41. Bruni, R., H. W. Taeusch, and A. J. Waring, Surfactant Protein B: Lipid interactions of synthetic peptides representing the amino-terminal amphipathic domain. Proc. Natl. Acad. Sci. USA, 1991. 88: p. 7451-7455.
42. Lipp, M. M., et al., Phase and morphology changes in lipid monolayers induced by SP-B protein and its amino-terminal peptide. Science, 1996. 273: p. 1196-1199.
43. Lipp, M. M., et al., Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers. Biophys. J., 1997. 72: p. 2783-2804.
44. Nag, K., et al., Phase transitions in films of lung surfactant at the air-water interface. Biophys. J., 1998. 74: p. 2983-2995.
45. Takei, T., et al., The surface properties of chemically synthesized peptides analogous to human pulmonary surfactant protein SP-C. Biol. Pharm. Bull., 1996. 19: p. 1247-1253.
46. Wang, Z., et al., Acylation of pulmonary surfactant protein-C is required for its optimal surface active interactions with phospholipids. J. Biol. Chem., 1996. 271: p. 19104-19109.
47. Simon, R. J., et al., Peptoids: A modular approach to drug discovery. Proc. Natl. Acad. Sci. USA, 1992. 89: p. 9367-9371.
48. Zuckermann, R. N., et al., Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid phase synthesis. J. Am. Chem. Soc., 1992. 114: p. 10646-10647.
49. Kruijtzer, J. a. L., R., Synthesis in Solution of Peptoids using Fmoc-protected N-substituted Glycines. Tetrahedron Letters, 1995. 36(38): p. 6969-72.
50. Miller, S. M., et al., Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid, and N-substituted gylcine peptide and peptoid oligomers. Drug Development Research, 1995. 35: p. 20-32.
51. Borman, S., Peptoids eyed for gene therapy applications. C & E News, 1998. 76: p. 56-57.
52. Kirshenbaum, K., et al., Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure. Proc. Natl. Acad. Sci., U.S.A., 1998. 95: p. 4303-4308.
53. Figliozzi, G. M., et al., Synthesis of N-substituted glycine peptoid libraries. Meth. Enzymology, 1996. 267: p. 437-447.
54. Curstedt, T., et al. Low molecular mass surfactant protein type I: The primary structure of a hydrophobic 8-kDa polypeptide with 8 half cystine residues. Eur. J. Biochem., 1988. 172: p. 521-525.
55. Johansson, J., T. Curstedt, and H. Jörnvall, Surfactant protein B: Disulfide bridges, structural properties, and kringle similarities. Biochemistry, 1991, 30: p. 6917-6921.
56. Johansson, J., H. Mrnvall, and T. Curstedt, Human surfactant polypeptide SP-B disulfide bridges, C-terminal end, and peptide analysis of the airway form. FEBS Lett., 1992. 301: p. 165-167.
57. Cochrane, C. G. and S. D. Revak, Pulmonary surfactant protein B (SP-B): Structure-function relationships. Science, 1991. 254: p. 566-568.
58. Van den Bussche, G., et al., Secondary structure and orientation of the surfactant protein SP-B in a lipid environment: A FTIR spectroscopy study. Biochemistry, 1992. 31: p. 9169-9176.
59. Pérez-Gil, J., A. Cruz, and C. Casals, Solubility of hydrophobic surfactant proteins in organic solvent/water mixtures: Structural studies on SP-B and SP-C in aqueous organic solvents and lipids. Biochim. Biophys. Acta, 1993. 1168; p. 261-270.
60. Johannson, J., et al., The NMR structure of the pulmonary surfactant-associated polypeptide SP-C in an apolar solvent contains a valyl-rich α-helix. Biochemistry, 1994. 33: p. 6015-6023.
61. Pastrana, B., A. J. Mautone, and R. Mendelsohn, FTIR studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids. Biochemistry, 1991. 30: p. 10058-10064.
62. Shiffer, K., et al., Lung surfactant proteins SP-B and SP-C alter the thermodynamic properties of the phospholipid membrane: A differential calorimetry study. Biochemistry, 1993. 32: p. 590-597.
63. Morrow, M. R., et al., 2H-NMR studies of the effect of pulmonary surfactant SP-C on the 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine headgroup: A model for transbilayer peptides in surfactant and biological membranes. Biochemistry, 1993. 32: p. 11338-11344.
64. Van den Bussche, G., et al., Structure and orientation of the surfactant-associated protein C in a lipid bilayer. Eur. J. Biochem., 1992. 203: p. 201-209.
65. Curstedt, T., et al., Hydrophobic surfactant-associated polypeptides: SP-C is a lipopeptide with two palmitoylated cysteine residues, whereas SP-B lacks covalently linked fatty acyl groups. Proc. Natl. Acad. Sci. USA, 1990. 87: p. 2985-2989.
66. Creuwels, L. A. J. M., et al., Neutralization of the positive charges of surfactant protein C: Effects on structure and function. J. Biol. Chem., 1995. 270: p. 16225-16229.
67. Johansson, J., Curstedt, T, Robertson, B, Synthetic protein analogues in artificial surfactants. Acta Paediatr, 1996. 85: p. 642-6.